Pharmaceutical composition and methods of uses

ABSTRACT

The present invention provides compositions or formulations comprising the combination of a compound of 5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione or a salt thereof or hydrate of the foregoing, and a cyclodextrin; or in further combination with an excipient comprising L-Arginine or other additives, wherein the compositions or formulations increases water solubility and oral bioavailability of 5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione and its related compounds. The method of making or using the compositions or formulations is also disclosed. Such compositions or formulations may be used in treatment of certain diseases including asthma through inhibition of matrix metalloproteinase-12.

This application claims priority from U.S. Provisional PatentApplication No. 62/377,427, filed on Aug. 19, 2017. The contents of theprovisional application are hereby incorporated into this application byreference.

FIELD OF THE INVENTION

The present invention relates to compositions of combination of5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione or itsanalogues with a cyclodextrin, and methods of use thereof. Thecompositions of5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione or itsanalogues with a cyclodextrin increase water solubility of5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione or itsanalogues thereby enhance the oral bioavailability. Moreover, thepresent invention relates to the use of pharmaceutical compositionscomprising 5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dioneor its analogues with a cyclodextrin in the treatment of a disease orcondition that is or is believed to be responsive to inhibition ofmatrix metalloproteinase-12.

BACKGROUND OF THE INVENTION

A series of compounds of5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione or itsanalogues were disclosed in United States Patent Application20060041000. These compounds were designed to be used as inhibitors ofmacrophage elastase. All these compounds are hydantoin derivatives andtested in vitro for their use as matrix metalloproteinase (MMP)inhibitors.

As shown in the United States Patent Application 20060041000, allcompounds tested show desirable activity and favorable selectivityprofile. IC50s on MMP-12 fall in the range of 1-300 nM, therefore theyare all considered to be active. Most of the above compounds do not showinhibition on MMP-1 and MMP-7 at 10 uM. Their selectivity for MMP-12over MMP-2, MMP-3, MMP-9 and MMP-13 range from 50 to 1000 fold. Thesecompounds seem to possess some potentials to be used in the treatment ofdiseases or conditions mediated by MMP-12, such as asthma, chronicobstructive pulmonary diseases (COPD), arthritis, cancer, heart diseaseand nephritis. However, except for the IC50 data for limited MMPs, therewere no further biological data provided in detail. In addition, exceptthe NMR and MS data, no other physical and chemical property data wereprovided.

Therefore, it would be desirable to understand the characteristics ofthese compounds and develop suitable formulations to use5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione or itsanalogues for potential treatment of various diseases through inhibitionof MMP.

According to the teaching of the United States Patent Application20060041000, MMP Inhibitory Assays were conducted in aqueous buffer (50mM Hepes, 10 mM CaCl₂), 0.05% Brij 35, pH 7.5), indicating that thesecompounds should be fairly water soluble. However, it was unexpectedlydiscovered that these compounds have very low aqueous solubility.5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione and itsanalogues not only exhibit a low solubility in water, but also in anacidic environment. Consequently, when administered orally in aconventional solid dosage form, a low bioavailability may be expected.

Therefore, there remains a need for developing formulations of thesecompounds, such as formulations that render these compounds suitable fornon-invasive such as oral, intranasal and/or sublingual administrations.

SUMMARY OF THE INVENTION

The present invention provides compositions or formulations comprisingthe combination of a compound of the formula (I) or a salt thereof orhydrate of the foregoing and a cyclodextrin.

Also provided are methods of producing compositions containing acompound of the formula (I) or a salt thereof and methods of treating adisease or indication that is responsive to a compound of the formula(I) or a salt thereof comprising administering a pharmaceuticalcomposition containing a compound of the formula (I) or a salt thereofto an animal or human subject.

One object of the present invention is to increase the water-solubilityof compound of the formula (I). The present invention thus relates tomethods for improving the solubility of compound of the formula (I),said method comprising a pharmaceutical composition comprising thecombination of a compound of the formula (I) and a cyclodextrin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Polarized Light Microscopy (PLM) results of raw material and theprepared complex of compound IVO:HPBCD (1:3, w/w) by spray dryingmethod;

FIG. 2 X-ray powder diffraction (XRPD) results of raw materials and theprepared complex of compound IVO:HPBCD (1:3, w/w) by spray dryingmethod;

FIG. 3 Effect of MBCD on the solubility of IVO in 2.5% L-ARG solution;

FIG. 4 Effect of HPBCD on the solubility of IVO in 2.5% L-ARG solution;and

FIG. 5 Effect of SBEBCD on the solubility of IVO in 2.5% L-ARG solution.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition comprising a compound ofthe formula (I) and a cyclodextrin. The composition significantlyincreases the solubility of the compound of the formula (I) in water.

According to the present invention, the compound of the formula (I),wherein R is selected from the group consisting of phenyl,4-benzyloxyphenyl, 4-biphenyl, 4-methoxyphenyl, 3-methoxyphenyl,2-methoxyphenyl, 3,5-dimethoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,2-chlorophenyl, 4-methylphenyl, 3-methylphenyl, 2-methylphenyl, and3-trifluoromethylphenyl.

It is understood that the salts, such as pharmaceutically acceptablesalts, and solvates thereof, are also intended by the descriptionsprovided herein. Thus, all salt and non-salt forms of the compound ofthe formula (I) and solvates of the foregoing are embraced by theinvention and descriptions of the compound of the formula (I) providedherein.

According to the present invention, the cyclodextrin for use in thecompositions herein is a water soluble unsubstituted or substitutedalpha-cyclodextrin (ACD), beta-cyclodextrin (BCD), or gamma-cyclodextrin(GCD). In some embodiments, the beta-cyclodextrin is selected from thegroup consisting of methyl beta-cyclodextrin (MBCD), hydroxypropylbeta-cyclodextrin (HPBCD), and sulfobutylether beta-cyclodextrin(SBEBCD). In some embodiments, the beta-cyclodextrin is methylbeta-cyclodextrin or hydroxypropyl beta-cyclodextrin. In someembodiments, the gamma-cyclodextrin is hydroxypropyl gamma-cyclodextrin(HPGCD). In one preferred embodiment, the cyclodextrin is hydroxypropylbeta-cyclodextrin (HPBCD) or methyl beta-cyclodextrin (MBCD).

According to the present invention, methods for improving the solubilityof a compound of the formula (I) in water comprising combining thecompound of the formula (I) with a cyclodextrin are provided. In oneembodiment, a method of increasing the water solubility of a compound offormula (I) is provided, wherein the method comprises forming aninclusion complex of a compound of the formula (I) and a cyclodextrin.In some embodiments, the solubility of the compound of the formula (I),when present as an inclusion complex with a cyclodextrin in deionizedwater at room temperature, is increased by at least 2-fold, whencompared to the solubility of the compound of formula (I) in anuncomplexed form under the same conditions. The term “room temperature”as defined herein is about 20 to 25 degrees Celsius with an average of23° C. In other embodiments, the solubility, such as the aqueoussolubility, of the compound of the formula (I) in a compositionincreases by at least 5-fold to 2,000-fold or more over compound of theformula (I) alone. Solubility comparisons may be assessed by methodsknown to one skilled in the art, such as any of the specific methods andconditions detailed herein.

According to the present invention, the oral bioavailability of thecompound of the formula (I), when present with a cyclodextrin, is atleast 50% greater than the oral bioavailability of the compound of theformula (I) in absence of a cyclodextrin. Oral bioavailability, andcomparisons thereof, may be assessed by methods known in the art,including any of the specific methods described herein.

According to the present invention, a composition of a compound offormula (I) and a cyclodextrin is provided, wherein the compositioninduces a greater maximum concentration (Cmax) of the compoundsystemically than what is achievable when the compound is administeredalone in the same amount and under the same conditions. In oneembodiment, a composition of the compound of formula (I) induces atleast 1.5 or more times greater Cmax for the compound systemically thanwhat is achievable when the compound is administered alone in the sameamount and under the same conditions. In some embodiments, the Cmax ofthe compound of the formula (I), when administered to an animal or humanwith a cyclodextrin, is at least 2 times greater than the Cmax of thecompound of the formula (I) administered alone under the sameconditions.

According to the present invention, a composition of a compound offormula (I) and a cyclodextrin is provided, wherein the compositioninduces a greater area under the plasma concentration vs. time curve(AUC) of the compound than what is achievable when the compound isadministered in the absence of a cyclodextrin in the same amount andunder the same conditions. In one embodiment, a composition of thecompound of formula (I) with a cyclodextrin induces at least 2 or moretimes greater AUC of compound than what is achievable when the compoundis administered in the absence of a cyclodextrin in the same amount andunder the same conditions. In some embodiments, the AUC of the compoundof the formula (I), when administered to an animal with a cyclodextrin,is at least 2 times greater than the AUC of the compound of the formula(I) administered in the absence of a cyclodextrin in the same amount andunder the same conditions.

According to the present invention, a composition of a compound offormula (I) and a cyclodextrin is provided, wherein the compositioninduces a change in the time to reach the maximum plasma level (Tmax) ofthe compound than what is achievable when the compound is administeredin the absence of a cyclodextrin in the same amount and under the sameconditions. In another embodiment, a composition of the compound offormula (I) with a cyclodextrin reduces the maximum plasma level (Tmax)of the compound by 1 or 2 fold over what is achievable when the compoundis administered in the absence of the cyclodextrin in the same amountand under the same conditions. In some embodiments, the Tmax of thecompound of the formula (I), when administered to an individual with acyclodextrin, is at least 2 times shorter than that of the compound ofthe formula (I) administered in the absence of the cyclodextrin in thesame amount and under the same conditions. In some embodiments, thecompound of the formula (I) with a cyclodextrin reduces Tmax by at leastany of 1, 2, and 3 hours or more.

According to the present invention, a composition comprising a compoundof the formula (I) and a cyclodextrin is provided, wherein the molarratio of the compound of formula (I) to the cyclodextrin is from 1:1 to1:300, preferably from 1:1 to 1:50, and more preferably from 1:1 to1:10. In one embodiment, the composition comprises a complex of acompound of the formula (I) and a cyclodextrin, wherein at least amoiety of compound of the formula (I) has inserted itself, at leastpartially, into the cavity of the cyclodextrin to form an inclusioncomplex. In another embodiment, the composition comprises a physicalmixture of a cyclodextrin and a compound of the formula (I), wherein thephysical mixture does not comprise or is substantially free of at leasta moiety of compound of the formula (I) has inserted itself, at leastpartially, into the cavity of cyclodextrin. In another embodiment, acomposition comprising a) a compound of the formula (I), or a saltthereof, or solvate of the foregoing; b) a cyclodextrin; and c) anadditive, are provided. In one embodiment, the additive is apharmaceutically acceptable excipient. In another embodiment, theadditive will further increase the solubility of compound of the formula(I) in an aqueous solution. The additive may be in liquid, solid orsemi-solid form. In some embodiments, the additive is selected from thegroup, but not limited to, consisting of citric acid, PEG-4000, PVP K40,PVP K10, NaCMC, L-Arginine, Lysine, and D-Mannitiol. In one preferredembodiment, the additive is L-arginine and in another preferredembodiment, the additive is lysine.

Compositions comprising a compound of the formula (I) and a cyclodextrinmay further comprise additional formulation components, also referred toherein as additional agents. In some embodiments of the formulationsdescribed herein, the formulation further comprises a carrier; in someother embodiments of the formulations described herein, the formulationfurther comprises an antioxidant.

According to the present invention, a composition comprising (a) acompound of the formula (I), or a salt thereof, or solvate of theforegoing; (b) a cyclodextrin; and (c) a carrier, is a solidformulation. In some embodiments, the formulation is a semi-solid. Insome embodiments, the formulation is a liquid.

EXAMPLES

The following examples illustrate the compositions and methods of thepresent invention. The examples do not limit the invention, but areprovided to teach how to make useful controlled release drug deliverycompositions.

Example 1: Synthesis of the Compound of Formula (I)

The synthesis of compound of the formula (I), i.e.,5-[3-(4-benzyloxyphenylthio)-fur-2-yl]-imidazolidin-2,4-dione and itsanalogues were performed according to the method disclosed in UnitedStates Patent Application 20060041000. The following compounds weresynthesized and characterized.

-   IVE: 5-{3-[4-(3-Methoxybenzyloxy)    phenylthio]fur-2-yl}imidazolidine-2,4-dione-   IVH: 5-{3-[4-(4-Chlorobenzyloxy)    phenylthio]fur-2-yl}imidazolidine-2,4-dione-   IVO: 5-{3-[4-(3-Methyl-benzyloxy)    phenylthio]fur-2-yl}imidazolidine-2,4-dione-   IVP: 5-{3-[4-(2-Methyl-benzyloxy)    phenylthio]fur-2-yl}imidazolidine-2,4-dione-   IVQ: 5-{3-[4-(3-Trifluoromethyl-benzyloxy)    phenylthio]fur-2yl}imidazolidine-2,4-dione

Example 2: Effect of Different Cyclodextrins on the Aqueous Solubilityof 5-{3-[4-(3-Methoxybenzyloxy)phenylthio]fur-2-yl}imidazolidine-2,4-dione (IVE)

Table 1 lists various commercially available cyclodextrins (CDs). Inorder to test the effect of various CDs on the aqueous solubility ofcompound IVE, 1 mL of each of the following aqueous solutions asdescribed in Table 2 was prepared. Excess IVE was added to each of thesesolutions and the samples were shaken at room temperature for 24 hourson an orbital shaker at 200 rpm. Excess IVE was present in all samplesat all times to obtain solubility. After 24-hours, the samples werecentrifuged. An aliquot of the supernatant was diluted, if necessary,and assayed by HPLC. Table 2 shows that both ACD and HPGCD do notimprove aqueous solubility of compound IVE significantly, while MBCD,SBEBCD, and HPBCD can significantly enhance the aqueous solubility ofcompound IVE. These results indicate that only beta cyclodextrins mayimprove the solubility of compound IVE. The size of the compound mayallow it to form an inclusion complex, resulting in a higher aqueoussolubility. The enhancement effect on the aqueous solubility of compoundIVO is in the order of MBCD>SBEBCD>HPBCD>HPGCD>ACD. MBCD is the mosteffective solubility improving agent in this experiment.

TABLE 1 Commercially available cyclodextrins Water Number of SolubilityCyclodextrin Glucopyranose Molecular (25° C.) Type Trade Name UnitsAbbreviation Weight (mg/mL) Methyl beta- CAVASOL ® 7 MBCD 1310 >750Cyclodextrin W7 M Pharma Hydroxypropyl KLEPTOSE ® 7 HPBCD 1400 >750beta-cyclodextrin HPB Pharma Sulfobutylether Captisol ® 7 SBEBCD2163 >500 beta-cyclodextrin Hydroxypropyl CAVASOL ® 8 HPGCD 1574 >750gamma- W8 HP Pharma cyclodextrin Alpha- CAVAMAX ® 6 ACD 973 145cyclodextrin

TABLE 2 Solubility of compound IVE in different CD's solutionConcentration of IVE (mg/mL) CD ACD Concen- Concen- tration HPBCD MBCDHPGCD SBEBCD tration ACD  0 mg/ml 0.023 0.023 0.023 0.023  0 mg/ml 0.023100 mg/ml 0.744 3.251 0.059 0.815 40 mg/ml 0.053

Example 3: Effect of Different Cyclodextrins on the Aqueous Solubilityof 5-{3-[4-(2-Methyl-benzyloxy)phenylthio]fur-2-yl}imidazolidine-2,4-dione (IVP)

In order to test the effect of various CDs on the aqueous solubility ofcompound IVP, 1 mL of each of the following aqueous solutions asdescribed in Table 3 was prepared. Excess IVP was added to each of thesesolutions and the samples were shaken at room temperature for 24 hourson an orbital shaker at 200 rpm. Excess IVP was present in all samplesat all times. After 24-hours, the samples were centrifuged. An aliquotof the supernatant was diluted, if necessary, and assayed by HPLC. Table3 shows that both ACD and HPGCD do not improve aqueous solubility ofcompound IVP significantly, while MBCD, SBEBCD, and HPBCD cansignificantly enhance the aqueous solubility of compound IVP. Theseresults indicate that only beta cyclodextrins may improve the solubilityof compound IVP. The size of the compound may allow it to form aninclusion complex, resulting in a higher aqueous solubility. Theenhancement effect on the aqueous solubility of compound IVP is in theorder of MBCD>SBEBCD>HPBCD>HPGCD>ACD. MBCD is the most effectivesolubility improving agent in this experiment.

TABLE 3 Solubility of compound IVP in different CD's solutionConcentration of IVP (mg/mL) CD ACD Concen- Concen- tration HPBCD MBCDHPGCD SBEBCD tration ACD  0 mg/ml 0.064 0.064 0.064 0.064  0 mg/ml 0.064100 mg/ml 0.727 6.269 0.001 0.805 40 mg/ml 0.015

Example 4: Effect of Different Cyclodextrins on the Aqueous Solubilityof5-{3-[4-(3-Methyl-benzyloxy)phenylthio]-fur-2-yl}-imidazolidin-2,4-dione(IVO)

In order to test the effect of various CDs on the aqueous solubility ofcompound IVO, 1 mL of each of the following aqueous solutions asdescribed in Table 4 was prepared. Excess IVO was added to each of thesesolutions and the samples were shaken at room temperature for 24 hourson an orbital shaker at 200 rpm. Excess IVO was present in all samplesat all times. After 24-hours, the samples were centrifuged. An aliquotof the supernatant was diluted, if necessary, and assayed by HPLC. Table4 shows that both ACD and HPGCD do not improve aqueous solubility ofcompound IVO, while MBCD, SBEBCD, and HPBCD can significantly enhancethe aqueous solubility of compound IVO. These results indicate that onlybeta cyclodextrins may improve the solubility of compound IVO. The sizeof the compound may allow it to form an inclusion body complex,resulting in a higher aqueous solubility. The enhancement effect on theaqueous solubility of compound IVO is in the order ofMBCD>SBEBCD>HPBCD>HPGCD>ACD. MBCD is the most effective solubilityimproving agent in this experiment.

TABLE 4 Solubility of compound IVO in different CD's solutionConcentration of compound IVO (mg/mL) CD ACD Concen- Concen- trationHPBCD MBCD HPGCD SBEBCD tration ACD  0 mg/ml 0.026 0.026 0.026 0.026  0mg/ml 0.026  25 mg/ml 0.037 0.244 0.009 0.094  5 mg/ml 0.037  50 mg/ml0.083 0.477 0.016 0.116 10 mg/ml 0.030  75 mg/ml 0.108 0.673 0.006 0.15615 mg/ml 0.026 100 mg/ml 0.184 1.066 0.002 0.174 20 mg/ml 0.013 150mg/ml 0.312 1.695 0.004 0.315 30 mg/ml 0.010 200 mg/ml 0.467 1.784 0.0030.761 40 mg/ml 0.017

Example 5: Effect of Different Cyclodextrins on the Aqueous Solubilityof 5-{3-[4-(3-Trifluoromethyl-benzyloxy)phenylthio]fur-2yl}imidazolidine-2,4-dione (IVQ)

In order to test the effect of various CDs on the aqueous solubility ofcompound IVQ, 1 mL of each of the following aqueous solutions asdescribed in Table 5 was prepared. Excess IVQ was added to each of thesesolutions and the samples were shaken at room temperature for 24 hourson an orbital shaker at 200 rpm. Excess IVQ was present in all samplesat all times. After 24-hours, the samples were centrifuged. An aliquotof the supernatant was diluted, if necessary, and assayed by HPLC. Table5 shows that both ACD and HPGCD do not improve aqueous solubility ofcompound IVQ significantly, while MBCD, SBEBCD, and HPBCD cansignificantly enhance the aqueous solubility of compound IVQ. Theseresults indicate that only beta cyclodextrins may improve the solubilityof compound IVQ. The size of the compound may allow it to form aninclusion body complex, resulting in a higher aqueous solubility. Theenhancement effect on the aqueous solubility of compound IVQ is in theorder of MBCD>SBEBCD>HPBCD>HPGCD>ACD. MBCD is the most effectivesolubility improving agent in this experiment.

TABLE 5 Solubility of compound IVQ in different CD's solutionConcentration of IVQ (mg/mL) CD ACD Concen- Concen- tration HPBCD MBCDHPGCD SBEBCD tration ACD  0 mg/ml 0.138 0.138 0.138 0.138  0 mg/ml 0.138100 mg/ml 1.215 5.776 N.D. 0.641 40 mg/ml 0.111

Example 6: Preparation of the Complex of Compound IVO with HPBCD bySolvent Evaporation Method

Approximate 10.0 g of compound IVO was weighed out into a volumetricflask and completely dissolved in 660 mL of methanol (MeOH) by usingultrasonication (15.2 mg/mL) to get a clear solution. After filtering itwith 0.45 um filter membrane to remove potential leftover crystal solid,approximate 30.05 g of HPBCD (Ashland) were added into the solution at aratio of 25:75 (w/w) of IVO:HPBCD. The sample was stirred for 60 min toform a clear solution before it was spray dried to obtain a soliddispersion. The sample obtained by spray drying was further dried undervacuum condition at 30° C. for 24 hrs. Based on Polarized LightMicroscopy (PLM) (FIG. 1) and X-ray powder diffraction (XRPD) (FIG. 2)results, the prepared complex of compound IVO:HPBCD (1:3, w/w) by spraydrying method was amorphous, and named as compound IVO/HPBCD ASD-01.FIG. 1 (left) shows that IVO alone is birefringent indicating that IVOis in crystal form. FIG. 1 (right) shows that the IVO/HPBCD complex doesnot exhibit any birefringence. FIG. 2 shows that the IVO/HPBCD complexdoes not exhibit any crystal diffraction signals. The IVO/HPBCD is mostlikely in the form of an inclusion complex, i.e., at least a moiety ofthe compound of the formula (I) has inserted itself, at least partially,into the cavity of the cyclodextrin. The formation of an inclusioncomplex changes IVO from crystal form to amorphous status.

TABLE 6 The details of the IVO/HPBCD ASD-01 Name ASD Formula IVO:HPBCD =25:75 (w/w) Weight 35 g white solid powder Yield (%) 87.5

TABLE 7 The spray drying parameter setting and results Parameterssetting Actual data Ratio (IVO:polymer, w/w) IVO:HPBCD: = 25:75 Nozzleorifice size (mm) 0.6 Set air speed (m³/min) 0.40 Actual air speed(m³/min) 0.40 Set inlet Temp. (° C.) 80 Actual air Temp. (° C.) 79.4Chamber out Temp. (° C.) 57.4 Cyclone in Temp. (° C.) 45.9 Upper chamber(mBar) 3.7 Cyclone pressure (mBar) 20.5 Set Nozzle airflow (L/min) 6.8Actual nozzle airflow (L/min) 7.6 Cyclone size medium Cooling air flow(m³/min) 0.1

Example 7: Comparable Solubility Test for IVO ASD-01

Excess IVO, 450 mg HPBCD, 150 mg IVO+450 mg HPBCD physical mixture and600 mg ASD-1 were prepared in 1.5 ml DI water, then those samples wereshaken at room temperature for 24 hours on an orbital shaker at 200 rpm.The samples were centrifuged at 3 and 24 hours. An aliquot of thesupernatant was diluted, if necessary, and assayed by HPLC. With sametesting condition, IVO ASD-01 showed much higher solubility in waterthan physical mixture (Table 8).

TABLE 8 Result of comparable solubility test 3 hrs 24 hrs pH atsolubility solubility 24 hrs Composition (mg/ml) (mg/ml) equilibrium 450mg HPBCD — — 7.06 Excess compound IVO 0.002 0.011 8.09 150 mg compoundIVO + 450 mg 2.400 2.207 6.88 HPBCD Physical Mixture 600 mg compound IVOASD-01 20.734 21.621 5.72

Example 8: Effect of Additional Excipient on the Solubility of IVO

This study was to explore if there is any synergistic effect by addingadditional excipients. As described in Table 9, each of the followingaqueous solutions was prepared containing 0; 0.125% 0.25%; 0.5%; 0.75%,1.5% and 2% additional excipient in 1 mL 30 mg/ml MBCD aqueous solution.An excess of compound IVO was added to each of these solutions and thesamples were shaken for 24 hours on an orbital shaker at 200 rpm and atroom temperature. Excess of compound IVO was present in all samples atall times. After 24-hours, the samples were centrifuged. An aliquot ofthe supernatant was diluted, if necessary, and assayed by HPLC. As shownin Table 9, only L-Arginine (L-ARG) shows significant solubilityimprovement of IVO in 30 mg/ml MBCD solution. The solubility of IVOdecreases with the increase of the concentration of citric acid andPEG-400, while no effect was observed for PVP K40, PVP K10, NaCMC, andD-mannitol.

TABLE 9 Excipient effect on the solubility of compound IVO in 30 mg/mlMBCD solution Excipient Concen- Solubility of compound IVO (mg/mL)tration Citric PEG- PVP PVP L- D- (w/v) Acid 4000 K40 K10 NaCMC ARGMannitiol   0% 0.18 0.18 0.18 0.18 0.16 0.18 0.18 0.125%  — — 0.20 0.180.17 0.70 0.20 0.25% 0.15 0.16 0.19 0.18 0.17 0.93 0.18 0.50% 0.13 0.150.18 0.18 0.16 1.24 0.18 0.75% 0.09 0.17 0.18 0.18 0.16 1.60 0.19   1%0.09 0.15 0.19 0.18 0.16 1.83 0.19 1.50% 0.07 0.15 0.19 0.18 0.18 2.060.18   2% 0.01 0.01 0.19 0.17 0.19 2.33 0.19

Example 9: Effect of L-Arginine on the Aqueous Solubility of CompoundIVO

1 mL of each of the following aqueous solutions was prepared containing0.15% to 2.5% L-ARG in DI water by weight. Excess compound IVO was addedto each of these solutions and the samples were shaken at roomtemperature for 24 hours on an orbital shaker at 200 rpm. Excesscompound IVO was present in all samples at all times. After 24-hours,the samples were centrifuged. An aliquot of the supernatant was diluted,if necessary, and assayed by HPLC. Table 10 shows that L-ARG only canimprove compound IVO aqueous solubility slightly, much less than thatobserved in Example 8. This indicates that there is a synergistic effectby combining MBCD and L-Arginine. One possibility is that L-Arginine mayhelp promote the formation of inclusion complex between IVO and MBCD.

TABLE 10 Solubility of compound IVO in solutions containing L-ArginineL-Arginine Compound IVO (% w/w) (ug/ml) 0 1.146 0.15 11.07 0.31 15.100.5 25.33 0.63 34.20 1.25 46.79 2.5 85.84

Example 10: Effect of CDs on the Solubility of Compound IVO in 2.5%L-ARG Solution

1 mL of each of the following aqueous solutions was prepared containing0; 25; 50; 75; 100; 150 and 200 mg/mL CD in 2.5% L-ARG solution. Anexcess of compound IVO was added to each of these solutions and thesamples were shaken at room temperature for 24 hours on an orbitalshaker at 200 rpm. Excess compound IVO was present in all samples at alltimes. After 24-hours, the samples were centrifuged. An aliquot of thesupernatant was, if necessary, diluted and assayed by HPLC. FIGS. 3, 4,and 5 shows that L-ARG exhibits syngeneic effect on solubilityimprovement with all 3 If-CD derivatives. The addition of L-Arginineenhanced the solubility of IVO in MBCD, HPBCD, and SBEBCD aqueoussolution for more than 5, 18, and 20 folds, respectively.

Example 11: A Pharmacokinetic Study of IVO Formulations in Beagle Dogs

Introduction

This example describes the pharmacokinetics (PK) of IVO after singleoral administration or intravenous injection of different formulationscontaining IVO to male Beagle dogs.

Materials

Test Animals:

Species: Dog; Strain: Beagle; Sex: male; Total Number: A total of 3males were selected for the study; Body Weight: 8 to 13 kg; Supplier:Marshalls BioResources (New York, N.Y., USA) All in-life records arekept at CTPS, QPS Taiwan.

Study Materials

Nonmicronized IVO Suspension

Nonmicronized IVO (PT-C12071028-F13001) was administered to animals viaoral administration as a suspension. The suspension was prepared in 10%(w/v) hydroxypropyl-β-cyclodextrin (HPBCD). A dose of 20 mg/kg was usedfor the 1^(st) dosing group.

Micronized IVO Suspension

Micronized IVO (D-1405FP1321-01) was administered to animals via oraladministration as a suspension. The suspension was prepared in 10% (w/v)HPBCD. A dose of 20 mg/kg was used for the 2^(nd) dosing group.

Nonmicronized IVO in Capsule

Nonmicronized IVO (PT-C12071028-F13001) was administered to animals viaoral administration as a capsule. Capsules were filled in gelatincapsules at testing facility on each dosing day. A dose of 20 mg/kg wasused for the 3^(rd) dosing group.

Micronized IVO in Capsule

Micronized IVO (D-1405FP1321-01) was administered to animals via oraladministration as a capsule. Capsules were filled in gelatin capsules attesting facility on each dosing day. A dose of 20 mg/kg was used for the4^(th) dosing group.

IV of Nonmicronized IVO

Nonmicronized IVO (PT-C12071028-F13001) was dissolved in DMSO andadministered to animals via IV injection. A dose of 4 mg/kg was used forthe 5 h dosing group

Methods

Study Design

Dosing and Blood Collection

This study was designed in a crossover fashion. Three male dogsconsecutively received single dose of Nonmicronized IVO Suspension (20mg/kg IVO, 1^(st) dosing), one dose of Micronized IVO Suspension (20mg/kg IVO, 2^(nd) dosing), one dose of Nonmicronized IVO in Capsule (20mg/kg IVO, 3^(rd) dosing), one dose of Micronized IVO in Capsule (20mg/kg IVO, 4^(th) dosing) and one dose of IV of Nonmicronized IVO (4mg/kg IVO, 5^(th) dosing) with a washout period (≥3 days) between eachtreatment in this pharmacokinetic study. Blood was collected from themale dogs pre-dose and at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hpost-dose from the animals in the 1^(st) dosing group, the 2^(nd) dosinggroup, the 3^(rd) dosing group and the 4^(th) dosing group, and pre-doseand at 5 min post-dose and 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hpost-dose from the animals in the 5^(th) dosing group into tubescontaining K₂EDTA as anticoagulant. Blood samples were placed on iceimmediately and centrifuged (1500×g for 10 minutes at 4° C.) within 60minutes of blood collection. Plasma samples were stored at −60° C. orbelow at CTPS, QPS Taiwan until transferred to QPS Taiwan. A summary ofdosing and blood collection times is shown in Table 11.

TABLE 11 Dosing and Blood Collection Dos- No. of Dose ing Male (mg/Blood Collection Day Treatment Type Animals^(a,b) Route kg) Times^(c) 1Nonmicronized 3 PO 20 Pre-dose, 0.25, 0.5, IVO Suspension 1, 2, 4, 6, 8,12 and 5 Micronized 3 PO 20 24 h post-dose IVO Suspension 11Nonmicronized 3 PO 20 IVO in Capsule 15 Micronized 3 PO 20 IVO inCapsule 20 IV of Non- 3 IV 4 Pre-dose, 5 min micronized IVO post-doseand 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post-dose ^(a)The same dogswere administered with different treatments in a crossover fashion witha washout period of more than 3 days between each dosing. ^(b)The animalIDs are 13M00001, 13M00007 and 13M00012. ^(c)Anticoagulant was K₂EDTA.

Plasma Bioanalytical Method

The plasma samples were analyzed at QPS Taiwan using a validatedLC-MS/MS method with an LLOQ of 5.000 ng/mL for IVO. Plasmaconcentrations below the lowest standard were reported as belowquantifiable limit (BQL).

Data Analyses

PK Parameters

PK parameters were determined at QPS Taiwan using non-compartmentalanalysis on individual profile using Phoenix® WinNonlin® 6.3 (PharsightCorporation, Mountain View, Calif., USA). The observed maximum plasmaconcentration (C_(max)) and the time of C_(max) (T_(max)) weredetermined directly from the data. The area under the plasmaconcentration-time curve from time-0 to 24 hour post-dose (AUC_(0-24 h))and the area under the plasma concentration-time curve from time-0extrapolated to infinity (AUC_(0-∞)) were determined by the lineartrapezoidal rule:

AUC_(0-24 h)=(t ₂ −t ₁)×(C ₁ +C ₂)/2

with extrapolation to infinity using:

AUC_(0-∞)=AUC_(last) +C _(last)/λ

Where possible, the apparent terminal elimination half-life (t_(1/2))was calculated according to the following formula, where λ is theterminal elimination rate constant:

t _(1/2)=ln(2)/λ

The selection criteria for inclusion of data points in the calculationof λ required that at least three data points representing the terminalphase were regressed and that r²≥0.85 when rounded. Half-life wasdefined as not determined (ND) if these criteria were not met. Totalclearance (CL), mean residence time extrapolated to infinity(MRT_(0-∞)), volume of distribution (V_(z)) and oral bioavailability (F)were determined according to the following formulae:

CL=Dose/AUC_(0-∞, IV)

MRT_(0-∞)=AUMC_(0-∞)/AUC_(0-∞)

V _(z)=Dose/(λ·AUC_(0-∞))

F=(AUC_(0-∞,po)/Dose_(po))/(AUC_(0-∞,IV)/Dose_(IV))

Nominal sample collection times were used for AUC, CL and t_(1/2)calculations. Nominal doses were used for dose normalized AUC_(0-∞) inall test article-treated groups.

Data Reporting Conventions

Plasma PK Data Analyses

Individual or mean plasma concentrations were reported to three decimalplaces. Mean plasma concentrations were calculated using SAS® andreported to three decimal places. Individual concentrations that wereBQL were set to zero for the calculation of PK parameters.

Individual plasma concentrations were input to WinNonlin™ using valuesto three decimal places. PK parameters with values up to 999 werereported to three significant figures and values ≥1000 were reported aswhole numbers, with the following exceptions:

T_(max) values are reported to one decimal place if the value is onehour or greater, and to two decimal places if the value is less than onehour.

λ values are reported to three decimal places.

CV % values are reported to one decimal place.

Rounding

The computer-generated data shown in the tables have been roundedappropriately for inclusion in this example. As a result, calculation ofvalues from data in this example, in some instances, yields minorvariations.

Results and Discussion

The dosing groups and the blood collection schedule for this study aresummarized in Table 11. PK parameters for all IVO-treated groups aretabulated in Table 12.

TABLE 12 PK Parameters of IVO in Male Dogs Following Single OralAdministration or IV Injection of Different IVO Formulations IVO PlasmaPharmacokinetic Parameters (N = 3) Nonmicronized MicronizedNonmicronized Micronized IV of IVO IVO IVO IVO Nonmicronized SuspensionSuspension in Capsule in Capsule IVO (20 mg/kg) (20 mg/kg) (20 mg/kg)(20 mg/kg) (4 mg/kg) Mean SD Mean SD Mean SD Mean SD Mean SD T_(max)_(a) (h) 4.0 (2.0-4.0) 4.0 (2.0-4.0) 4.0 (4.0-4.0) 1.0 (0.50-2.0) 0.08(0.08-0.08) C_(max) (ng/mL) 1037 198 2524 223 200 147 272 35.9 6818 1362AUC_(0-24 h) 13105 3227 29085 4014 1807 1648 1922 130 26959 5191 (h ·ng/mL) AUC_(0-∞) 15876 6692 35021 9135 1808 1696 1993 200 27856 5988 (h· ng/mL) λz (1/h) 0.118 0.064 0.095 0.040 0.215 0.026 0.149 0.033 0.1520.029 t_(1/2) (h) 7.6 5.1 8.2 3.3 3.3 0.4 4.8 1.2 4.7 1.0 MRT_(0-∞) — —— — — — — — 6.6 1.2 (h) Vz (L/kg) — — — — — — — — 0.972 0.059 CL — — — —— — — — 0.148 0.028 (L/h/kg) F (%)^(b) 11.1 2.79 25.1 4.00 1.43 1.451.46 0.23 — — —: Not applicable. ^(a) Median (range). ^(b)F(%) wascalculated as (AUC_(0-∞, po)/Dose_(po))/(AUC_(0-∞, IV)/Dos)

A total of 3 male Beagle dogs were dosed in a cross-over design withfive IVO formulations with at least a 3-day washout period between eachdosing according to the protocol. No incident with potentiallysignificant impact on the outcome of the study was observed.

No quantifiable levels of IVO were found in all pre-dose samples.Following oral administration of Nonmicronized IVO Suspension (1^(st)dosing group), Micronized IVO Suspension (2^(nd) dosing group),Nonmicronized IVO in Capsule (3^(rd) dosing group) and Micronized IVO inCapsule (4^(th) dosing group), C_(max) of IVO were observed within2.0-4.0 h, 2.0-4.0 h, all 4 h and 0.5-2.0 h, respectively. Mean C_(max)values were 1037, 2524, 200 and 272 ng/mL in the 1^(st), 2^(nd), 3^(rd)and 4^(th) dosing groups, respectively.

Mean AUC_(0-24 h) values of IVO were 13105, 29085, 1807 and 1922h·ng/mL, and mean AUC_(0-∞) values of IVO were 15876, 35021, 1808 and1993 after oral administration in the 1^(st), 2^(nd), 3^(rd) and 4^(th)dosing groups, respectively. Plasma levels declined with a t_(1/2) of7.6, 8.2, 3.3 and 4.8 h in the 1^(st), 2^(nd), 3^(rd) and 4^(th) dosinggroups, respectively (Table 12).

Following IV bolus dosing of Nonmicronized IVO at the dose levels of 4mg/kg, C_(max) of IVO was observed at the first sampling time, 0.08 hpost-dose, and averaged 6818 ng/mL. Mean AUC_(0-24 h) and AUC_(0-∞)values of IVO were 26959 and 27856 h·ng/mL, respectively. Plasma levelsdeclined with a t_(1/2) of 4.7 h. Mean systemic clearance was 0.148L/h/kg and mean Vz was 0.972 L/kg. Mean MRT_(0-∞) was 6.6 h (Table 12).Following oral administration of Nonmicronized IVO Suspension,Micronized IVO Suspension, Nonmicronized IVO in Capsule and MicronizedIVO in Capsule, the oral bioavailability (F) of IVO was 11.1%, 25.1%,1.43% and 1.46%, respectively.

Conclusions

Animals were dosed with IVO as intravenous or oral formulation by singleadministration. Study results showed that the absorption rate and drugexposure of IVO in the suspension-treated groups were greater than thosein the capsule-treated groups. Following oral administration ofNonmicronized IVO Suspension, Micronized IVO Suspension, NonmicronizedIVO in Capsule and Micronized IVO in Capsule, the oral bioavailability(F) was 11.1%, 25.1%, 1.43% and 1.46%, respectively

Example 12: A Parallel Relative Oral Bioavailability Study of IVO OralFormulations Following Single Oral (PO) Administration to Non-Naïve MaleBeagle Dogs

Objective of the Study

The objective of this study was to determine the relative oralbioavailability of two IVO formulations (filled in capsules and dosed at150 mg/dog) compared to a reference formulation of nonmicronized IVOsuspension at 20 mg/kg after single oral administration to non-naïvemale beagle dogs. Plasma samples were collected from all animals for upto 24 hours post dose, and concentration of IVO was determined byLC-MS/MS method.

Animal Welfare

All applicable portions of the study conformed to the followingregulations and guidelines regarding animal care and welfare:

AAALAC International and NIH guidelines as reported in the “Guide forthe Care and Use of Laboratory Animals,” National Research Council—ILAR,Revised 2011. People's Republic of China, Ministry of Science &Technology, “Regulations for the Administration of Affairs ConcerningExperimental Animals”, 1988.

Materials and Methods

Test Article and Reference Standard Information

Test formulations and reference standard powder were supplied by theSponsor. The details and certificate of analysis (CoA) are listed asbelow:

Molecular Molecular Storage Weight in Weight in Purity Condi- CompoundID Batch No. Free Form Salt Form (%) tions IVO-d9 QP111102 403.50403.50 >99 RT (Internal Standard) IVO PT- 394.44 394.44 98.9 RT(Bioanalytical C12071028- Standard) F13001

Molecular Molecular Weight in Weight in Salt Purity Compound ID BatchFree Form Salt Form Factor* (%) IVO (Group 1, PT- 394.44 394.44 1.0098.9 non-micronized C12071028- API) F13001 *Salt Factor = Molecularweight in salt form/Molecular weight in free form

Potency (%) (% Purity Correct Formulation ID Batch drug load) (%) FactorIVO ASD-01 YF00892-003-141219-01 24.19 99.20 4.167 (Group 2) IVOgranules YF00892-006-141224-01 23.81 99.01 4.242 (Group 3) CorrectFactor = (Molecular weight in salt form/Molecular weight in freeform)/Purity/Potency

Test System, Study Design and Animal Care

Test System

Nine non-naïve male beagle dogs (7.40-9.78 kg) were used in this study.Animals were obtained from an approved vendor (Marshall Bioresources,Beijing, China) and each animal had a unique skin tattoo number on earas the identification.

Study Design

Target Target Dose Target Dose # of Animal Dose Dose VolumeConcentration Group Males ID Test Article Formulation Route Level(mL/kg) (mg/mL) 1 3 D101 IVO Suspension (reference) PO 20 mg/kg 4.0 5.0D102 suspension Vehicle: 10% (w/v) Hydroxypropyl- D103 β-cyclodextrin(HPBCD) in water for injection 2 3 D201 IVO Capsule (test) PO 150 mg/dogNA NA D202 ASD-01 D203 3 3 D301 IVO Capsule (test) PO 150 mg/dog NA NAD302 granules D303

Animal Care

The animal room was controlled and monitored for humidity (targeted meanrange 40% to 70%) and temperature (targeted mean range 18° C. to 26° C.)with 10 to 20 air changes/hour. The room was on a 12-hour light/darkcycle except when interruptions were necessitated by study activities.

Animals were individually housed in stainless-steel mesh cages duringin-life that were in accordance with the National Research Council“Guide for the Care and Use of Laboratory Animals”

Animals were fed twice each day. Stock dogs were fed approximately 220grams of Certified Dog Diet daily (Beijing Vital Keao Feed Co., Ltd.Beijing, P. R. China). These amounts were adjusted as necessary based onfood consumption of the group or an individual body weight changes ofthe group or an individual and/or changes in the certified diet.

For fasted group (PO dose groups) animals were fed the afternoon (at3:30 to 4:00 pm) prior to the day of oral dosing and the remaining foodwas removed at about 7:00 pm. Food was withheld until 4-hour post-doseunless specified in this protocol. Fasted animals were fed once on theday of dosing, with the amount of approximately 220 grams.

Animals were provided reverse-osmosis purified and chlorinated water adlibitum by an automated watering system.

Nutritional components and environmental contaminants in the diet wereanalyzed routinely by the vendor or an independent laboratory,respectively. The analysis reports and lot numbers are on file at theTesting Facility.

The animal drinking water was analyzed for contaminants each quarter byan independent laboratory. Water analysis reports are on file inVeterinary Operation's department at the Testing Facility.

Formulation Preparation

For suspension formulation preparation:

IVO suspension for Group 1 at a concentration of 5 mg/mL in 10% (w/v)Hydroxypropyl-β-cyclodextrin in water for injection was prepared on thedosing day. The detailed information of the vehicle used and the doseformulation preparation procedure were recorded in the study folder.

For capsule formulation preparation:

Gelatin capsules (size: 0#) were used in this study for Groups 2 & 3.

-   -   a. Animals were weighed on the dosing day, and the body weight        ranging at 10±1 kg were selected, except for 7.91 kg of animal        D303 was selected.    -   b. 150 mg API of the formulation was filled in the capsules.    -   c. 3 capsules in total were dosed per dog.

Details of capsules weight are presented in Table 13.

TABLE 13 Capsule weights Capsule Drug Animal Weight Total Test ArticleRoute Group No. (mg) (mg) IVO ASD-01 PO 2 D201 212.19 625.11 in capsule202.77 210.15 D202 211.31 625.05 204.84 208.90 D203 209.47 625.13 208.33207.33 IVO Granules 3 D301 212.09 636.39 in capsule 214.13 210.17 D302216.87 636.30 213.64 205.79 D303 206.79 637.51 207.84 222.88

Administration

The IVO formulations were orally administered in accordance with SOPs:

Orally for suspension formulation:

The gavage doses were flushed using 6 mL of vehicle (approximately 3times volume of gavages tube). All tubes were equal size and cut toequal length so that the flush volume was comparable.

Orally for capsule formulation:

-   -   a. Pulled lower jaw of dogs down and placed the capsule far back        in the throat. The capsule was then pushed past the pharynx by        using a thumb or index finger.    -   b. To facilitate swallowing of capsule, 6 mL of water was given        to the animal following each capsule administration.    -   c. After administering the capsule, swallowing was induced by        gently stroking the dog's throat.    -   d. After administration, the dog's mouth was inspected to ensure        that the capsule had been swallowed.

Animals were weighed prior to each dose administration, and the bodyweights of the individual animals are presented in Table 14.

TABLE 14 Animal Body Weights Body Drug Animal Weight Test Article RouteGroup No. (kg) IVO (non- PO 1 D101 8.63 micronized D102 8.77 API) D1037.40 IVO ASD-01 2 D201 9.35 in capsule D202 9.08 D203 9.78 IVO Granules3 D301 9.29 in capsule D302 9.33 D303 7.91

Sample Collection and Preparation

Serial blood samples (approximately 0.8 mL in 10 μL 0.5M K2-EDTA) werecollected via venipuncture from a cephalic vein from non-sedated animalsat pre-dose (0), 0.25 (15 mins), 0.5 (30 mins), 1, 2, 4, 6, 8, 12 and 24hours post dose. Actual sample collection times were recorded in thestudy folder. For samples collected within the first hour of dosing, a±1 minute of the scheduled time was acceptable. For the remaining timepoints, samples that were taken within 5% of the scheduled time wereacceptable and were not considered as protocol deviation.

After collection, blood samples were gently inverted several times andimmediately placed on wet ice prior to centrifugation at 2-8° C. and3000×g for 10 minutes within 1 hour after blood collection. The plasmasamples were then transferred into labeled polypropylenemicro-centrifuge tubes, snap frozen and transferred to bioanalysisdepartment on dry ice, and stored frozen in a freezer set to maintain−60° C. or lower until bio-analysis.

Clinical Observation

Twice daily (at approximately 9:30 a.m. and 3:30 p.m.), animals wereobserved for mortality and signs of pain and distress. Cage-sideobservations for general health and appearance were made once daily. Onthe dosing day, the animals were observed before and after each bloodcollection time point. Any unusual observations noted throughout theduration of the study were recorded in the study folder.

Sample Analysis

Plasma samples were analyzed by using a LC/MS-MS method. The lower limitof quantification (LLOQ) for IVO in plasma was 2.00 ng/mL and the upperlimit of quantification (ULOQ) was 3000 ng/mL.

Pharmacokinetic Data Analysis

Plasma concentration-time profiles of IVO were subjected to anon-compartmental pharmacokinetic analysis using WinNonlin softwareprogram (version 6.2.1).

The mean residence time (MRT), the area under the plasma concentrationtime curve (AUC) from time zero to the last quantifiable time point(AUC0-last) and AUC from time zero to infinity (AUC0-inf) werecalculated using the linear-log trapezoidal rule (See: Gabrielsson J.and Weiner D. Non-compartmental analysis in “Pharmacokinetic andPharmacodynamic Data Analysis: Concepts & Applications”, 3rd edition,Chapter 3.7.2., page 141-146. Swedish Pharmaceutical Press; 2002).

All the values except for time values were reported to three significantfigures. Time values were reported to two decimal places.

Nominal sampling times were used to calculate all pharmacokineticparameters since there was no deviation between the actual and thenominal sampling times.

Results

Clinical Observations

About 10 mL light yellow vomited mucus with empty capsule shell wasobserved for animal D203 of Group 2 at 1 hour post dose. No abnormaleffect was observed for the other study animals during this study.

Dose Concentration Verification

Dose concentration verification of Group 1 showed an accuracy of 101%.

Pharmacokinetics

Pharmacokinetic parameters of IVO are presented in Table 15.

TABLE 15 Pharmacokinetic parameters of IVO formulations IVO IVO IVOASD-01 Granules suspension (Capsule, (Capsule, Formulation (20 mg/kg)150 mg/dog) 150 mg/dog) Dose (mg/kg) 20.2 16.0 17.1 C_(max) (ng/mL) 14533947 1071 T_(max) (h) 4.00 1.67 3.33 AUC_(0-last) (ng · h/mL) 1263020100 7077 AUC_(0-inf) (ng · h/mL) 12963 20233 6215 AUC_(0-last)/Dose625 1249 410 (ng · h/mL/(mg/kg)) MRT_(0-last) (h) 6.66 4.52 7.17MRT_(0-inf) (h) 7.20 4.65 5.82 Relative F % (based NC 200 65.5 onAUC_(0-last))

Conclusions

Following single oral administration of non-micronized IVO suspension at20 mg/kg to non-naïve male beagle dogs in fasted state, the maximumplasma concentration (Cmax) was 1453±90.7 ng/mL occurred at 4.00±3.46hours post dose (Tmax). The plasma exposures, AUC0-inf and AUC0-lastwere 12963±4191 and 12630±3948 ng·h/mL, respectively.

Following single oral administration of IVO ASD-01 in capsule at 150mg/dog to non-naïve male beagle dogs in fasted state, the maximum plasmaconcentration (Cmax) was 3947±1740 ng/mL occurred at 1.67±0.58 hourspost dose (Tmax). The plasma exposures, AUC0-inf and AUC0-last were20233±8545 and 20100±8391 ng·h/mL, respectively.

Following single oral administration of IVO granules in capsule at 150mg/dog to non-naïve male beagle dogs in fasted state, the maximum plasmaconcentration (Cmax) was 1071±597 ng/mL occurred at 3.33±2.31 hours postdose (Tmax). The plasma exposures, AUC0-inf and AUC0-last were 6215±NCand 7077±3749 ng·h/m L, respectively.

Compared with the reference formulation, IVO ASD-01 in capsule wasabsorbed more rapidly (Tmax=1.67), while IVO granules in capsules showeda similar Tmax at 3.33 hours compared to the reference formulation. Thesystemic exposure (AUC) and maximum plasma concentrations (Cmax) of IVOwere much higher for IVO ASD-01 in capsule than those of the referenceformulation. The IVO granules in capsule showed a lower oral absorptioncompared to the reference formulation.

The relative bioavailability of IVO ASD-01 and IVO granules was 200% and65.5% compared with the reference formulation, respectively.

Example 13: Comparison of IVO/HPBCD ASD-in-Capsule to IVO API-in-Capsulein Beagle Dogs

A formulation of nonmicronized IVO oral suspension (20 mg/kg) was usedas a reference. The suspension was prepared in 10% (w/v)hydroxypropyl-β-cyclodextrin (HPBCD). A dose of 20 mg/kg was used (IVOSuspension) as shown in Examples 11 and 12.

IVO API-in-Capsule was tested in Example 11 with the reference.IVO:HPBCD complex at a ratio of 25:75 (w/w) was prepared according tothe procedure described in Example 6. IVO/HPBCD ASD-in-Capsule wastested in Example 12 along the reference.

Table 16 shows the PK parameters of three different formulations dosedorally in beagle dogs. IVO API is highly crystalline and has very lowaqueous solubility. The formulation IVO API-in-Capsule (neat API filledin oral capsules) showed lower AUC and Cmax than that of the referenceformulation. In contrast, an improved oral formulation, IVO/HPBCDASD-in-Capsule, shows significantly increased aqueous solubility. As aresult, the oral absorption (AUC) and Cmax of IVO was significantlyincreased using IVO/HPBCD complex ASD-in-Capsule, by approximately17-fold and 18-fold, respectively, in dogs when compared to IVOAPI-in-Capsule, as shown in Table 16.

TABLE 16 PK comparison of IVO/HPBCD ASD-in-Capsule to IVO API-in-Capsulein Beagle Dogs Study Example 11 Example 12 Formulation Test ReferenceTest Reference Description API-in- IVO ASD-in- IVO Capsule suspensionCapsule suspension Dose (mg/kg) 20.0 20.0 16.0 20.2 C_(max) (ng/mL) 2001037 3947 1453 T_(max) (h) 4.0 4.0 1.7 4.0 AUC_(0-inf) 1808 15876 2023312963 (h*ng/mL) AUC_(0-inf)/Dose 90.4 793.8 1264.6 641.7 C_(max)/Dose10.0 51.9 246.7 71.9 AUC_(0-inf) Ratio 17 (ASD/API) ^(a) C_(max) Ratio18 (ASD/API) ^(a) ^(a) Ratios were calculated after normalization withthe parameters of reference formulation.

Compared with the reference formulations, IVO/HPBCD ASD-01 in capsulewas absorbed more rapidly (Tmax=1.7), while IVO API-in-Capsule showed asame Tmax at 4 hours in comparison to the reference formulation.Overall, HPBCD can significantly improve the solubility of IVO in waterand enhance systemic absorption.

1. A pharmaceutical composition comprising a compound of the formula (I) or a salt thereof or hydrate of the foregoing; and a cyclodextrin, wherein R is selected from the group consisting of phenyl, 4-benzyloxyphenyl, 4-biphenyl, 4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl, 3,5-dimethoxyphenyl, 4-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl, 4-methylphenyl, 3-methylphenyl, 2-methylphenyl, and 3-trifluoromethylphenyl.


2. The pharmaceutical composition of claim 1, wherein the molar ratio of the compound of formula (I) to the cyclodextrin is from 1:1 to 1:50.
 3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition increases the solubility of the compound of formula (I) in deionized water at room temperature, by at least 1.5-fold compared to the solubility of the compound of formula (I) without the cyclodextrin under the same conditions.
 4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition increases the oral bioavailability of the compound of formula (I) in a mammal by at least 50% over the oral bioavailability of the compound of formula (I) without cyclodextrin.
 5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition induces at least 2-times greater Cmax of the compound of formula (I) than what is achievable when the compound is administered alone in the same amount and under the same conditions.
 6. The pharmaceutical composition of claim 1, wherein the cyclodextrin is a water soluble unsubstituted or substituted alpha-cyclodextrin, beta-cyclodextrin, or gamma-cyclodextrin.
 7. The pharmaceutical composition of claim 6, wherein the beta-cyclodextrin is selected from the group consisting of methyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and sulfobutylether beta-cyclodextrin.
 8. (canceled)
 9. The pharmaceutical composition of claim 1, wherein at least a moiety of the compound of the formula (I) has inserted itself, at least partially, into the cavity of the cyclodextrin.
 10. The pharmaceutical composition of claim 1, wherein the composition comprises a physical mixture of the compound of the formula (I) and the cyclodextrin.
 11. A pharmaceutical composition comprising a compound of the formula (I) or a salt thereof or hydrate of the foregoing; a cyclodextrin; and an excipient, wherein R is selected from the group consisting of phenyl, 4-benzyloxyphenyl, 4-biphenyl, 4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl, 3,5-dimethoxyphenyl, 4-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl, 4-methylphenyl, 3-methylphenyl, 2-methylphenyl, and 3-trifluoromethylphenyl.


12. The pharmaceutical composition of claim 11, wherein the excipient is L-Arginine.
 13. The pharmaceutical composition of claim 11, wherein the molar ratio of the compound of formula (I) to the cyclodextrin is from 1:1 to 1:50.
 14. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition increases the solubility of the compound of formula (I) in deionized water at room temperature, by at least 1.5-fold compared to the solubility of the compound of formula (I) without the cyclodextrin under the same conditions.
 15. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition increases the oral bioavailability of the compound of formula (I) in a mammal by at least 50% over the oral bioavailability of the compound of formula (I) without cyclodextrin.
 16. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition induces at least 2-times greater Cmax of the compound of formula (I) than what is achievable when the compound is administered alone in the same amount and under the same conditions.
 17. The pharmaceutical composition of claim 11, wherein the cyclodextrin is a water soluble unsubstituted or substituted alpha-cyclodextrin, beta-cyclodextrin, or gamma-cyclodextrin.
 18. The pharmaceutical composition of claim 17, wherein the beta-cyclodextrin is selected from the group consisting of methyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, and sulfobutylether beta-cyclodextrin.
 19. (canceled)
 20. The pharmaceutical composition of claim 11, wherein the excipient is selected from the group consisting of L-Arginine, Citric Acid, PEG-4000, PVP K40, PVP K10, NaCMC, and D-Mannitol.
 21. The pharmaceutical composition of claim 11, wherein at least a moiety of the compound of the formula (I) has inserted itself, at least partially, into the cavity of the cyclodextrin.
 22. The pharmaceutical composition of claim 11, wherein the composition comprises a physical mixture of the compound of the formula (I), the cyclodextrin, and the excipient.
 23. A pharmaceutical composition comprising a compound of 5-{3-[4-(3-Methyl-benzyloxy) phenylthio]fur-2-yl}imidazolidine-2,4-dione (IVO); and a cyclodextrin, wherein the cyclodextrin is hydroxypropyl beta-cyclodextrin.
 24. The pharmaceutical composition of claim 23, wherein the weight ratio of the compound of formula (I) to the cyclodextrin is 1:3.
 25. A pharmaceutical composition comprising a compound of 5-{3-[4-(3-Methyl-benzyloxy) phenylthio]fur-2-yl}imidazolidine-2,4-dione (IVO); a cyclodextrin; and an excipient, wherein the cyclodextrin is hydroxypropyl beta-cyclodextrin; and the excipient is L-Arginine. 